Molecular Cancer Therapeutics 1069 Structure-based design of a selective heparanase inhibitor as an antimetastatic agent Keisuke Ishida,1,2 Go Hirai,3 Koji Murakami,2 Introduction 1,4 1 Takayuki Teruya, Siro Simizu, h D 3 1,4 Heparanase is an endo- - -glucuronidase that degrades Mikiko Sodeoka, and Hiroyuki Osada heparan sulfate glycosaminoglycans (1). Human hepara- 1Antibiotics Laboratory, RIKEN Discovery Research Institute, nase cDNA encodes the latent enzyme, which consists of Saitama, Japan; 2Hanno Discovery Center, Taiho Pharmaceutical 543 amino acid residues, and becomes the active enzyme of Co., Ltd., Saitama, Japan; 3Institute of Multidisciplinary Research 50 kDa after being processed twice at the amino terminus for Advanced Materials, Tohoku University, Miyagi, Japan; and (2, 3). Because only one heparanase cDNA sequence coding 4 Graduate School of Science and Engineering, Saitama University, functional enzyme has been identified to date, heparanase Saitama, Japan has been considered a major enzyme that degrades heparan sulfate glycosaminoglycans in mammalian tissues (4–7). High-level expression of heparanase enzyme has been Abstract found in highly invasive normal and malignant cells, such Heparanase is an endo-hhhhhh-D-glucuronidase that degrades as activated immune cells, lymphoma, melanoma, and car- heparan sulfate glycosaminoglycans in the extracellular cinoma cells (2, 8–11), as well as in human head and neck matrix and the basement membrane and is well known to tumors (12). Further, heparanase expression has been cor- be involved in tumor cell invasion and angiogenesis. We related with the metastatic property of tumor cells (2, 10, have focused on heparanase as a target for antitumor 11, 13) and experimental metastasis in an animal model agents, especially antimetastatic agents. (R)-3-hexadeca- was shown to be reduced by treatment with heparanase noyl-5-hydroxymethyltetronic acid (RK-682) was found to inhibitors (14, 15). It has also been shown that patients with display an inhibitory activity against heparanase in our a high-level expression of heparanase mRNA tend to have screening of natural sources. Because RK-682 has been a shorter postoperative survival term (16, 17). Heparanase reported to show inhibitory activities against several also enhances angiogenesis by the release of growth factors, enzymes, we have tried to develop selective heparanase such as basic fibroblast growth factor and vascular endo- inhibitors using the method of rational drug design. Based thelial growth factor, which are sequestered by heparan on the structure of the heparanase/RK-682 complex, we sulfate glycosaminoglycans in the extracellular matrix (10, speculated that selective inhibitory activity against hepar- 18–20). From these results, heparanase has been assumed anase could be acquired by arylalkylation, namely, by ben- to be a promising target for antitumor agents, especially zylation of the 4-position of RK-682. Among the rationally antimetastatic agents. designed 4-alkyl-RK-682 derivatives, 4-benzyl-RK-682 Most of the heparanase inhibitors that have been re- has been found to possess a selective inhibitory activity ported are sulfated oligosaccharide derivatives that resem- for heparanase (IC50 for heparanase, 17 Mmol/L; IC50 for ble the substrate of heparan sulfate glycosaminoglycans other enzymes, >100 Mmol/L). 4-Benzyl-RK-682 also (15, 21–23). We have established a heparan sulfate degra- inhibited the invasion and migration of human fibrosarco- dation assay system to develop novel heparanase inhibitors ma HT1080 cells (IC50 for invasion, 1.5 Mmol/L; IC50 for that are not sulfated oligosaccharide derivatives and have migration, 3.0 Mmol/L). On the other hand, RK-682 had no used it to screen 10,000 microbial broths of actinomycetes, inhibitory effect on the invasion and migration of HT1080 fungi, and bacteria (24). As a result, (R)-3-hexadecanoyl-5- cells at doses of up to 100 Mmol/L. [Mol Cancer Ther hydroxymethyltetronic acid (RK-682; Fig. 1A), which we 2004;3(9):1069–77] had purified previously as a potent inhibitor of tyrosine phosphatase (25), was found to possess a potent inhibitory activity for heparanase (24). The IC50 value of RK-682 for heparanase was 17 Amol/L; this is a fairly low value compared with, for example, the IC50 of suramin, a known heparanase inhibitor, which is 46 Amol/L (26). Received 12/26/03; revised 5/18/04; accepted 7/6/04. RK-682 has already been shown to have several Grant support: Ministry of Education, Science, Sports, Culture and inhibitory activities, including phospholipase A2 inhibition Technology of Japan and the Chemical Biology Project (RIKEN). (IC50,16Amol/L; ref. 27), HIV-1 protease inhibition (IC50, The costs of publication of this article were defrayed in part by the A payment of page charges. This article must therefore be hereby marked 84 mol/L; refs. 28, 29), and protein tyrosine phosphatase advertisement in accordance with 18 U.S.C. Section 1734 solely to inhibition (IC50 of CD45, 54 Amol/L; IC50 of vaccinia H1- indicate this fact. related phosphatase, 2.0 Amol/L; ref. 25). In the present Requests for reprints: Hiroyuki Osada, Antibiotics Laboratory, RIKEN study, we therefore attempted to develop a heparanase Discovery Research Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Phone: 81-48-467-9541; Fax: 81-48-462-4669. selective inhibitor based on rational drug design using RK- E-mail: [email protected] 682 as a lead structure and investigated the antitumor Copyright C 2004 American Association for Cancer Research. activity. Mol Cancer Ther 2004;3(9). September 2004 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2004 American Association for Cancer Research. 1070 A Selective Heparanase Inhibitor carried out by the Biopolymer and Discover3 modules of Discover/InsightII program (Accelrys) using the X-ray structure of 1,4-h-xylanase (Protein Data Bank entry 1BG4) as a template (31). Synthesis of 4-Methyl-RK-682 4-Methyl-RK-682 (4-Me-RK-682) was prepared as de- scribed previously (32). RK-682 was treated with diazo- methane in diethyl ether at 23jC for 20 minutes. Synthesis of 4-Isopropyl-RK-682 1,2,3-Triisopropyl-isourea was prepared as described previously (33). A solution of RK-682 (30.0 mg, 81.4 Amol) and 1,2,3-triisopropyl-isourea (30.3 mg, 162.8 Amol) in 0.5 mL dry tetrahydrofuran was stirred under reflux for 18 hours. After cooling to room temperature, the solution was concentrated. The residue was purified by silica gel col- umn chromatography (hexane/ethyl acetate, 5:1) and pre- parative thin-layer chromatography on silica gel (hexane/ ethyl acetate, 1:1) to give 4-isopropyl-RK-682 (4-iPr-RK- 24 682, 6.8 mg, 20%): [a]D =5.76(c =0.20,CHCl3); Fourier transformation-IR (film) 3,440, 2,917, 2,850, 1,722, 1,672, 1,603, 1,466, 1,409, 1,317, 1,097, 1,048 cmÀ1; 1 y H nuclear magnetic resonance (400 MHz, CDCl3) 5.38 (1H, septet, J = 5.6 Hz), 4.70 (1H, triplet, J = 3.2 Hz), 4.05 (1H, multiplet), 3.85 (1H, multiplet), 3.00 (1H, double triplet, J = 18.0, 7.8 Hz), 2.94 (1H, double triplet, J = 18.0, 7.8 Hz), 1.87 (1H, broad), 1.57 (2H, multiplet), 1.25 (22H, multiplet), 0.87 (5H, multiplet); 13C nuclear magnetic y resonance (100 MHz, CDCl3) 197.7, 177.2, 105.3, 79.6, 78.7, 61.5, 42.5, 31.9, 29.9, 29.68, 29.66, 29.62, 29.52, 29.48, 29.4, 29.2, 27.4, 23.8, 22.7, 22.1, 21.8, 14.1; matrix- associated laser desorption/ionization time of flight mass spectroscopy (positive ion, a-cyano-4-hydroxycynnamic + acid) calculated for C24H42O5Na: (M + Na) 433.293; found 433.235. Figure 1. Speculated molecular basis of the interaction between Synthesis of 4-Benzyl-RK-682 heparanase and ligands. A, structures of RK-682 and its 4-alkyl 2-Benzyl-1,3-dicyclohexyl-isourea was prepared as de- derivatives. B, interaction between the active site of heparanase and an scribed previously (33). A solution of RK-682 (30.0 mg, heparan sulfate disaccharide, GlcA-GlcNAc. C, interaction between the active site of heparanase and RK-682. Heparan sulfate, RK-682, active- 81.4 Amol) and 2-benzyl-1,3-dicyclohexyl-isourea (51.2 mg, site Glu residues, and the amino acid residues that interacted with ligands 162.8 Amol) in 0.5 mL dry tetrahydrofuran was stirred are in boldface type. Atoms in heparan sulfate and RK-682 are in green under reflux for 24 hours. After cooling to room tem- (carbon), red (oxygen), and blue (nitrogen). Atoms in heparanase are in white (carbon), red (oxygen), blue (nitrogen), and light blue (hydrogen). perature, the solution was concentrated. The residue was Yellow arrows, enzyme reaction of Glu225 (proton donor) and Glu343 purified by silica gel column chromatography (hexane/ (nucleophile). Blue dashed lines, hydrogen bonds. ethyl acetate, 5:1) and preparative thin-layer chroma- tography on silica gel (hexane/ethyl acetate, 1:1) to give 24 4-benzyl-RK-682 (4-Bn-RK-682, 20.1 mg, 54%): [a]D = À0.25 (c = 0.75, CHCl3); Fourier transformation-IR (film) 3,422, 2,918, 2,850, 1,766, 1,607, 1,433, 1,368, 1,329, 1,232, 1,075 À1 1 y Materials and Methods cm ; H nuclear magnetic resonance (400 MHz, CDCl3) Homology Modeling of Heparanase and Construction 7.41-7.30 (5H, multiplet), 5.50 (1H, doublet, J = 12.0 Hz), of the Heparanase/RK-682 Complex 5.34 (1H, doublet, J = 12.0 Hz), 4.78 (1H, triplet, J = 3.2 Hz), The amino acid sequence alignment of human heparanase 4.08 (1H, broad multiplet), 3.90 (1H, broad multiplet), 2.87 and 1,4-h-xylanase from Penicillium simplicissimum (EC (1H, double triplet, J = 17.8, 7.6 Hz), 2.78 (1H, double 3.2.1.8) was carried out manually using the Homology triplet, J = 17.8, 7.6 Hz), 2.04 (1H, broad), 1.51 (2H, mul- module of the Discover/InsightII programs (Accelrys, Inc., tiplet), 1.25 (22H, multiplet), 0.86 (5H, multiplet); 13C nu- y SanDiego,CA).Structurally conserved regions were defined clear magnetic resonance (100 MHz, CDCl3) 197.8, 177.5, by referring to the results of database searches and site- 170.3, 134.2, 129.1, 128.8, 128.0, 106.9, 78.7, 77.4, 61.2, directed mutagenesis by Hulett et al.